RPKM with bowtie output

Question

[flogin]

Hello guys,

There is a way to evaluate RPKM direct with bowtie 2 output (e.g. bam or sorted bam)? like an R package or some scripts to do this?

[ATpoint]

Is this RNA-seq? Are you aware bowtie2 is not splice-aware? What organism? Do you have a GTF file or list of reference peaks (if not RNA-seq)?

[flogin]

It's a mapping of RNA-seq against a set of contigs, I just have the contigs, rna-seq files and bam files.

Answer 1

[swbarnes2]

RSEM will take a bam file as input and return raw counts, RPKM and TPM

[flogin]

thanks swbarnes2, I'll check this.

[anoo]

Hello, Can you please provide the steps used for calculating expression using RSEM, after building the reference. The script is throwing errors.

[ATpoint]

Please read the manual and then come back with more specific questions while opening a new question, providing the necessary details.

[WouterDeCoster]

The script is throwing errors.

How do you expect us to help if you don't tell us what's going wrong?

[anoo]

I used the same commands given in the manual, with the BAM files I have,

software/RSEM-1.2.25/rsem-calculate-expression -p 8 --paired-end \ --bam \ --estimate-rspd \ --append-names \ --output-genome-bam \ exp/LPS_6h.bam \ ref/mouse_ref exp/LPS_6h

Error:

Warning: The SAM/BAM file declares less reference sequences (955) than RSEM knows (41078)! Please make sure that you aligned your reads against transcript sequences instead of genome. RSEM can not recognize reference sequence name 1!

"rsem-parse-alignments /home/user/data/RSEM/Rat /home/user/data/RSEM/Rat.temp/Rat /home/user/data/RSEM/Rat.stat/Rat /home/ec2-user/data/Rat/*.bam 3 -tag XM" failed! Plase check if you provide correct parameters/options for the pipeline!